The present invention relates to mouse mammary adenocarcinoma cell lines expressing estrogen and progesterone receptors as a tool to study the effect of hormones, pharmacological compounds, and environmental agents. The present invention also relates to methods in vitro and in vivo for testing hormones or other related molecules by cell proliferation or tumor proliferation.
Epithelial breast malignant neoplasms of the mammary gland is one of the most common form of cancer among women in North America, South America, Europe, and Australia, and they account for 15-18% of the deaths in this population (Lynn and Reiss, 1995 J. Natl Cancer Inst. 87: 867; Moller Jensen et al., 1990 Eur. J. Cancer 26: 1167-1256; Boring et al., 1993 Cancer J. Clin. 43: 7-26). Despite the development of different therapeutic approaches, the mortality has continued to rise over the past thirty years: the incidence of breast cancer is increasing by about 1% per year in almost all populations in both industrialized and developing countries (Miller et al., 1991 Cancer Causes Control 2:67-74; Levi F., 1993 Eur J Cancer 29:2315-2319), and it is estimated that the disease will affect 5 million women in the next decade (Corry J F. and Lonning P E., 1994 Pharmacol. Econom 5: 198-212). Obviously, within the aging female population, prevention and treatment of breast cancer will continue to represent a major challenge.
The etiology of breast cancer remains largely unknown, and the dilemma of mammary tumor development and the related mechanisms have been studied with different experimental approaches. The virus-induced mouse mammary tumor (MMTV) model had different drawbacks; most of the tumors induced by MMTV are not hormone responsive or are pregnancy-responsive (Welsch C W and Nagasawa H., 1977 Cancer Res 37:951-963; Sluyser M. and Van Nie R., 1974 Cancer Res 34:3253-3257), and no definite involvement was demonstrated for a virus in the etiology of breast tumors in humans (Michalides R. et al., Current Topics in Microbiology and Immunology, Vol. 106, Eds: P K Vogt y H Koprowski, Springer Verlag, Berlin, pp57-78; Pogo B G. et al., 1997 Medicina (Buenos Aires)57 Suppl 2:75-80). The chemical carcinogen models allowed the dissection of initiators and promoters; the tumors originated in both the MNU and the DMBA rat models were hormone-responsive, and they expressed estrogen receptors (ER) (Gullino P. et al., 1975 J Natl Cancer Inst. 54:401-404; Russo I H. et al., 1982 Breast Cancer Res and Treat 2:5-73). They did not, however, give rise to metastasis, and they harbored point mutations in certain oncogenes not found in the human disease that are probably related to the chemical carcinogen (Ip C., 1996 J. Mammary Gland Biol Neopl 1: 37-47).
Other approaches involve the establishment of human cell lines. The most widely used human breast cancer cell lines are the MCF-7 (Soule H D. et al., 1973 J Natl Cancer Inst 51:1409-1416); the T-47-D (Engel L W. et al., 1978 Cancer Res. 38: 3352-3364); and the ZR-75-31 (Keydar I. et al., 1979 Cancer 51: 659-670). These lines express ER and progesterone receptors (PR) and are hormone responsive. In the last years, other cell lines have been established: PMC42 (Whitehead R H. et al., 1984 J National Cancer Institute 73:643-648); YMB-1 (Yamane M. et al., 1984 Hiroshima J Med Sci 33:715-720); VHB (Vandewalle B. et al., 1987 J Cancer Res Clin Oncol 113:550-558); IBEP-1, IBEP-2 and IBEP-3 (Siwek B. et al., 1998 Int J Cancer 76:677-683); BrCa-MZ-01 and BrCa-MZ-02 (Mobus V J. et al., 1998 Int J Cancer 77:415-423). A part of our knowledge on hormone regulation of cell growth has been deduced from experiments performed using these lines. Approximately twenty cell lines, which express hormone receptors, are available and have been used in other types of experiments. It is well-known that valuable initial information can be gathered from in vitro studies. However, cancer occurs in the context of a complex interaction with its surrounding environment, e.g., neighboring tissues, the immune system, hormones, and environmental factors. Thus, there is a trend to study either xenografts in immune-suppressed mice (Clarke R. 1996 Breast Cancer Res and Treat 39: 69-86) or transgenic or knock-out mice (Amundadottir L T. Et al. 1996 Breast Cancer Res and Treat 39:119-135). A problem with most cell lines is that they are not metastatic unless they are transfected with different growth factors, and also the high costs of immuno-suppressed mice. In mice there are few models which have been used to study hormone regulation in tumor growth, among than them MXT model and the GR mice. The former is a mammary tumor which is maintained by syngeneic transplantation and expresses high levels of ER and PR (Kiss R. et al. 1989 Cancer Res 49:2945-2951). The latter is a E) strain of mice which develops pregnancy-dependent tumors (Sluyser M. and Van Nie R. 1974 Cancer Res 34:3253-3257). No cell lines have yet been developed from mouse tumors which maintain steroid receptor expression except for a tumor induced in c-erbB2 transgenic mice (Sacco M G. Et al 1998 Breast Cancer Res and Treat 47:171-180).
The inventors have developed an experimental model in which ductal metastatic, progestin-dependent (PD) mammary adenocarcinomas are induced by the continuous administration of MPA to BALB/c female mice; these tumors express high levels of ER and PR (Lanari C. et al 1986 Cancer Letters 33: 215-223., Molinolo A A. et al 1987 JNCI 79:1341-1350), and are maintained through syngeneic serial passages in MPA-treated mice. By transplantation into untreated mice, the inventors have been able to generate progestin-independent (PI) tumor lines that retain the expression of ER and PR. In in vitro studies, using primary and secondary cultures of one of these PD tumor lines (C4-HD), the inventors were able to demonstrate that MPA stimulates directly cell proliferation and that antiprogestins inhibit cell growth even at very low concentrations (Dran G. et. al. 1995 Breast Cancer Res and Treat 35: 173-186).
Earlier in the characterization of the experimental model, with the aim of further dissecting certain aspects of the hormonal response, the inventors developed a technique to obtain purified epithelial or fibroblastic primary cultures from these tumors (Dran G. et al. 1995 Breast Cancer Res and Treat 35:173-186). Although primary cultures are an excellent tool to study the direct effect of hormones on cell proliferation, the approach is time consuming, e.g., many different controls need to be performed in parallel, to be able to bypass the inherent heterogeneity of every primary culture, and standardize the findings.
To overcome these shortcomings, in the setting of the characterization of the different isoforms of the PR expressed by the tumors, the inventors decided to attempt to develop continuous cell lines in which different parameters regarding hormone dependence could be studied. The inventors were able to develop four cell lines derived from one PD tumor and one from a PI tumor. This is the first description of mouse mammary adenocarcinoma cell lines obtained in non transgenic animals which express ER and PR.
The inventors provided a cell line panel for screening hormones and related molecules.
In accordance with the present invention, cell lines and methods for screening hormones are provided.
The present invention relates two mouse mammary adenocarcinoma cell lines MC4-L1 and MC4-L3, which are derived from a murine progestin-dependent CC4-HD tumor, wherein the cell lines express ER and PR and are deposited in the American Type Culture Collection (ATCC) as Accession number PTA-889 and PTA-891.
The present invention also relates a cell line, MC4-L2, obtained by subcloning of cell line MC4-L1 deposited with the ATCC as Accession number PTA-892
The present invention also relates to a mouse mammary adenocarcinoma cell line MC7-L1 which is derived from the murine progestin-independent C7-H1 tumor, wherein the cell line expresses ER and PR and have been deposited on Oct. 28, 1999 with American Type Culture Collection (ATCC), 1081 University Blvd, Manassas, Va. 20110-2209 ATCC Accession number PTA-890.
A further object of the invention is to provide a cell culture system that will facilitate the screening and identification of agents that may be clinically relevant in the treatment of cancer. This cell line system will also find utility in the identification of compounds that have therapeutic value for other illnesses, e.g., endocrine disorders and other hormone-replacement therapies. A cell line system is provided for identifying, detecting or quantitating substances suspected of binding ER and PR.
The cell culture system of the present invention will be useful as a research tool rand will facilitate the elucidation of the mechanistic action of novel substances that bind ER and PR.
The present invention provides an in vitro method for testing the activity of hormones, anti-hormones, pharmacological compounds and environmental agents on cell lines. This method comprises exposing the cell lines MC4-L1, MC4-L3, MC4-L2 and MC7-L1 to hormones, anti-hormones, pharmacological compounds and environmental agents and then evaluating the cell proliferation by using any cellular proliferation method known by those skilled in the art. This includes 3H-thymidine uptake, MTT assay, MTS assay, and the like thereof.
The present invention also provides an in vivo method for testing the activity of hormone, anti-hormone, pharmacological compounds and environmental agents by inoculating cell lines MC4-L1, MC4-L3, MC4-L2 and MC7-L1 in syngeneic mice, allowing the tumors to grow to about 50 mm2 and applying a dose of the compounds to be evaluated daily and then analyzing the tumor size (tumor growth), tumor regression, number of metastases, prolongation of survival or any other parameter known to the experts in order to determine the activity of molecules tested on the tumor.
The cell lines and methods of the present invention provide a system for the analysis of hormones and related molecules without the need for primary tissue cultures.
It is an object of this invention to provide a kit that is highly useful for testing the activity of hormones, anti-hormones, pharmacological compounds and environmental agents. The kit comprises an aliquot of cell lines MC4-L1, MC4-L3, MC4-L2 and MC7-L1 and a method to evaluate cellular proliferation.
As described herein, the term xe2x80x9cagents that may be clinically relevant in the treatment of cancerxe2x80x9d comprises compounds or molecules that elicit a proliferative response through binding the PR or ER, such as, but not limited to, medroxyprogesterone acetate or R5020, diethylstylbestrol.
As described herein, xe2x80x9canti-hormonexe2x80x9d comprises molecules or compounds acting by inhibiting the proliferative activity of the above mentioned compounds, for example, but not limited to, mifepristone, ZK or tamoxifen, and raloxifene.
As described herein, xe2x80x9ccell linexe2x80x9d comprises cells that initially derived from a tumor. Such cells have indefinite growth in culture; unlike primary cultures, which can be maintained only for a finite period of time. Moreover, such cells preferably can form tumors after they are injected into syngeneic animals.
According to the invention xe2x80x9cCancerxe2x80x9d includes cancers, in particular breast cancer.
As described herein, xe2x80x9ccellular proliferationxe2x80x9d means an increasing in the cell number analyzed by any method known in the art, for example 3H-thymidine uptake, MET assay, MTS assay, relative to an untreated control.
As described herein, xe2x80x9ctumor growthxe2x80x9d includes an increase in tumor size and xe2x80x9ctumor regressionxe2x80x9d includes a reduction in tumor mass.
As described herein, xe2x80x9ctumor linexe2x80x9d includes tumors that express high levels of ER and PR, and are maintained through syngeneic serial passages in MPA-treated mice or into untreated mice.
As described herein, RU is mifepristone or RU38486; ZK is onapristone or ZK 98299.